This proposal examines two aspects of transcription by RNA polymerase II with a focus on events that occur at and around the TATA element. We will I) investigate the role of TFIIA in transcriptional processes of activated transcription during the response to oxidative stress; and 2) continue our functional studies of the factors involved in post-recruitment steps of transcription. To accomplish these aims, a combination of genetic, molecular and biochemical approaches will be employed in Saccharomyces cerevisiae. Studies proposed in Specific Aim 1 are based on our observation that mutant forms of TFIIA are defective for response to the yeast transcriptional activator Yap 1. Yap 1 is the key regulator of oxidative stress in yeast, and the response to oxidative stress is conserved from yeast to humans. In fact, oxidative stress appears to play a role in a variety of human conditions, including neurodegenerative diseases, cancer, and aging. As such, we propose a thorough biochemical and genetic characterization of the TFIIA-Yapl connection. These results will certainly advance our understanding of the mechanism of activation of genes required for protection from oxidative damage. Studies described in Specific Aim 2 are focused on events that occur in transcriptional regulation after TBP is recruited to the promoter. Post-recruitment step(s) could involve targeting of a necessary factor to the promoter, or alterations in interactions such that promoter melting, promoter clearance, or elongation efficiency are impacted. We have developed a novel genetic screen for identifying and classifying gene products involved in post-recruitment steps of transcription. One such gene, SPN1 is essential and highly conserved throughout evolution. As such, this specific aim describes studies for a further characterization of SPN1 (using genetic and biochemical techniques). We will also identify additional components that can influence post-recruitment steps of transcriptional regulation. Results generated from this proposal will provide important information on the factors and mechanisms essential for transcriptional regulation directly relevant to the human condition, since the processes of transcriptional control are conserved from yeast to humans.